Time-resolved spectroscopy is a widely used technique in the Biological sciences. In fluorescence spectroscopy, light at a specific frequency is absorbed by a given molecule or fluorescent entity (also called fluorophore), exciting its electronic state. The fluorescent entity then emits light at a slightly different frequency, as the fluorophore returns to the original ground state. Fluorescence spectroscopy is analogous to Raman spectroscopy in that a pump light excitation induces the emission of Stokes light, shifted to a lower frequency relative to the pump light. However, fluorescence requires the absorption of the pump light of a specific frequency, the frequency depending on the electronic system of the fluorescent entity. Also, contrary to Raman scattering, typical Stokes shifts for fluorescence phenomena are a few 10's of nm apart from the pump light, which complicates the cross-talk between the pump light and the Stokes signals at the detection level. Furthermore, as opposed to Raman scattering, which is essentially instantaneous, fluorescence emission takes place across a wide range of lifetimes, within a few ns or up to a few ms, depending on the fluorophore.
There are other types of time-resolved spectroscopy techniques widely used in biological applications and trace-gas sensing, such as Cavity Ring-Down Spectroscopy (CRDS).
In general, time-resolved spectroscopy techniques are mostly limited to laboratory environments due to the following reasons:
1) Short lifetime measurement techniques require the use of expensive and delicate equipment: pulsed pump lasers and state-of-the-art synchronized photo-detection schemes.
2) Time-resolved spectroscopy instrumentation is bulky due to the use of specially aligned optics, and high-end, photo-detector arrays.
3) Conventional time-resolved techniques such as fluorescence require the use of high performance optical filters to mitigate the cross-talk between pump and Stokes fluorescence signals, between the Stokes signals from different fluorophores, or the absorption signal of different molecules. This adds to the cost of the instrument and its complexity, reducing the signal collection efficiency.
4) Due to the extra complexity and cross-talk added by the optical filtering procedures, only small number of target substances can be analyzed simultaneously (3 or 4 at a time).
5) In fluorescence lifetime measurements, fluorophore concentration values are normally disregarded, as the measurement technique is only involved with relative changes of the signal in time. Also, the analytical complexity of deriving both lifetime and concentration values increases rapidly with the number of targets being analyzed. As a result, current lifetime fluorescent techniques are limited to fixed concentration measurements for a few target substances (2, 3 or 4).
6) Due to the close spectral proximity between the pump and Stokes signals in fluorescence spectra, and between Stokes signals from different fluorophores, high-performance optical filtering techniques are required. This increases cost and complexity of typical fluorescence devices.
In view of the above, there is a need for a time-resolved spectroscopy system that can be implemented in field applications under harsh environmental conditions. These applications usually require measurement of multiple targets (10 to 25) simultaneously. A complete measurement and sample assessment needs to be performed in a time frame of 1 s or less. Such a device would not only find new applications but also enhance current technologies like DNA sequencing and fluorescence imaging microscopy.